1. Field of the Invention
The present invention relates to a method of secretory production (producing and secreting) of heterologous proteins in coryneform bacteria, and more particularly, to a method of secretory production of heterologous proteins, including industrially useful enzymes and physiologically active proteins, in coryneform bacteria.
2. Brief Description of the Background Art
Coryneform bacteria are extremely useful bacteria in the fermentation industry as producers of L-amino acids, such as L-glutamic acid and L-lysine, as well as nucleic acids. In addition, coryneform bacteria inherently secrete extremely low levels of proteins extracellularly as compared with molds, yeast, and Bacillus species bacteria. Bacillus are typically preferable when secreting heterologous proteins, since purification of the heterologous proteins can be simplified or abbreviated. Coryneform bacteria also grow rapidly in a simple medium containing sugars, ammonia, and inorganic salts, making them superior in terms of cost, culturing method, and culture productivity, and are considered to be extremely useful bacteria in the production of heterologous protein as well.
Examples of methods for efficiently producing and secreting heterologous proteins using coryneform bacteria include secretion of nuclease and lipase by Corynebacterium glutamicum (to be abbreviated as C. glutamicum) (U.S. Pat. No. 4,965,197, J. Bacteriol., 174, 1854-1861 (1992)), secretion of protease such as subtilisin (Appl. Environ. Microbiol., 61, 1610-1613 (1995)), secretion of cellular surface layer protein of coryneform bacteria (Japanese International Patent Application Laid-open No. H6-502548), secretion of fibronectin-bound protein using coryneform bacteria (Appl. Environ. Microbiol., 63, 4392-4400 (1997)), secretion of a protein using a mutant secretion component (Japanese Patent Application Laid-open No. H11-169182), producing and secreting transglutaminase (Appl. Environ. Microbiol., 69, 358-366 (2003)), and producing and secreting transglutaminase using a mutant strain (WO 02/81694). In terms of the amount of protein able to be produced, accumulation of about 2.5 mg/ml of protein has been observed in C. glutamicum when expressing an alkaline protease gene derived from Dichelobacter nodosus using a promoter of the subtilisin gene (aprE) derived from Bacillus subtilis, a ribosome binding site, and a signal peptide sequence (Appl. Environ. Micribiol., 61, 1610-1613 (1995)). Regarding the secretion of transglutaminase, a a maximum amount of 930 mg/L has been confirmed (WO 02/81694).
The previously known pathway of protein secretion in coryneform bacteria is the pathway known as the Sec system (machine). The Sec machine is present in inner cytoplasmic membranes, and is composed of components primarily containing SecY (Japanese Patent Application Laid-open No. H6-169780), SecE (Japanese Patent Application Laid-open No. H6-277073), and SecG (Japanese Patent Application, Laid-open No. H11-169182), which function as protein secretion channels, and SecA (Japanese Patent Application Laid-open No. H7-107981) which functions as the driving force for protein permeation. This system is present in a wide range of microorganisms, ranging from prokaryotes including Escherichia coli and Bacillus subtilis, to eukaryotes including yeasts, molds, and humans, and is the most important and most common protein secretion pathway.
However, it is difficult to secrete some proteins using the Sec system in coryneform bacteria, and examples of such proteins include industrially useful proteins such as isomaltodextranase and protein transglutaminase.
A protein secretion pathway which is completely different from the Sec system was recently discovered in the thylakoid membrane of plant cell chloroplasts (EMBO J., 14, 2715-2722 (1995)). An arginine-arginine sequence is common to the signal sequences of proteins secreted through this pathway (EMBO J., 14, 2715-2722 (1995)), and as a result, this pathway has come to be referred to as the Tat system (Twin-Arginine Translocation system). Subsequently, this Tat system was determined to be specifically involved in the secretion of proteins having the common arginine-arginine signal sequence such as E. coli reductokinase, nitrate reductase, Bacillus subtilis lipoic acid synthetase, and phosphodiesterase (Science, 278, 1467-1470 (1997), U.S. Pat. Nos. 6,022,952, 6,335,178, J. Biol. Chem., 275, 41350-41357, International Patent Publication No. WO 02/22667).
In addition, while a protein is secreted before folding in the Sec system, the Tat system is characteristic in that a folded protein is secreted through the cell membrane (J. Biol. Chem., 25, 273(52), 34868-74 (1998)).
Although genes which have high homology with the genes encoding the Tat system components are present in coryneform bacteria as well, including tatA (GENEBANK cg103060 1571065-1571382), tatB (GENEBANK cg103060 1167110-1167580), tatC (GENEBANK cg103060 1569929-1570873), and tatE (gi|41223046|emb|CAF18991.1|), their functions are not known, and it is not known whether proteins are secreted by the Tat system pathway in coryneform bacteria.
In addition, although there are reports of improved secretion into the periplasm using the Tat pathway when introducing a plasmid which expresses the tatA, tatB, and tatC genes into E. coli, only about 5 to 10 mg/L cells was produced, which is not an industrially practical level (Biochem. Biophys. Res. Commun., 304, 279-284 (2003)).